It is proposed to: 1) establish the SN2-general acid catalysis mechanism for beta-glucuronidase obtained from mammalian tissue and to identify catalytic residues involved; 2) study effects of substituents in p-substituted phenyl S-beta-D-glucuronides on rate constants for glycosyl enzyme formation with a view toward understanding the nature of the transition state for reaction; 3) synthesize and test prototype reversible and irreversible inhibitors beta-glucuronidase inhibitors; 4) test for the selective accumulation of 6-mercaptopurine in tumor tissue (vs. normal tissue) of test rats fed glucose prior to, and during dosing with 6-mercaptopurine beta-D-glucuronide. These objectives shall be attacked by use of presteady state and steady state kinetics methods which shall involve stopped-flow and conventional spectrophotometry and use of group specific protein reagents: the combined chemical and kinetics approach is intended to reveal quantitatively mechanism and identity of catalytic amino acid participants. Design and synthesis of active site directed irreversible inhibitors and reactions of beta-glucuronidase with them should contribute to the above goal as well as provide information useful in designing an isoenzyme-specific inhibitor of beta-glucuronidase of potential therapeutic utility. Testing for the selective accumulation of 6-mercaptopurine in tumor tissue shall be accomplished by extraction of animal tissue and testing quantitatively via chromatographic and spectrophotometric techniques for 6-mercaptopurine.